Project Summary The central theme of this proposal is structural and mechanistic studies of nucleic acids, proteins, and their interactions. The two broad topics that will serve as foci for our research are: 1) the structure and mechanism of topoisomerases, and 2) the structure, architecture, and mechanism of RNase P. The aim of our work is to discover and explore paradigms that help to illuminate the structure and mechanism of biological molecules common to all three domains of life (bacteria, archaea, and eukaryota). Topoisomerases are ubiquitous proteins found across all three domains of life. They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. The study of the structure, mechanism, and function of topoisomerases not only furthers our understanding at many different levels of proteins that interact with DNA and alter its topological properties, but also provides important information to aid in the design of new therapeutic agents. To accomplish our goals, our comprehensive studies of topoisomerases will focus on two large and complementary projects: Project 1. Structural and biochemical studies of type IA, type IC and type II topoisomerases, and Project 2. Single molecule studies of type IA and type II topoisomerases. These two major projects will help answer crucial questions that are needed to advance the field to the next level. RNase P is a ribonucleoprotein complex found in almost all organisms, from bacteria to humans, and responsible for processing many different RNA molecules in the cell. It is composed of one essential RNA subunit and one or more proteins, but in all cases the RNA component is responsible for catalysis. RNase P was one of the first catalytic RNA molecules discovered and its study has been pivotal to our understanding of the role of RNA molecules in catalysis. Our studies of the structure and function of RNase P are providing important and relevant information on a key ribozyme involved in RNA processing in the cell, while also furthering our understanding of the structure and function of large RNA molecules and ribonucleoprotein complexes. To ensure that RNase P continues to be a paradigm for understanding RNA structure and function, large ribonucleoprotein complexes, and also the evolution from the ancient RNA world, a third project, Project 3. Structural and biophysical studies of RNase P, will be centered on structural and functional studies on RNase P expanding it to include RNase Ps from all three domains of life.